Pontibacillus sp. ALD_SL1 and Psychroflexus sp. ALD_RP9, two novel moderately halophilic bacteria isolated from sediment and water from the Aldabra Atoll, Seychelles

Pontibacillus sp. ALD_SL1 and Psychroflexus sp. ALD_RP9 are two novel bacterial isolates from mangrove sediment and a moderately hypersaline pool on the Aldabra Atoll, Seychelles. The isolates represent two novel species were characterised physiologically and genomically. Pontibacillus sp. ALD_SL1 is a facultatively anaerobic yellow, motile, rod-shaped Gram-positive, which grows optimally at a NaCl concentration of 11%, pH 7 and 28°C. It is the third facultatively anaerobic member of the genus Pontibacillus. The organism gains energy through the fermentation of pyruvate to acetate and ethanol under anaerobic conditions. The genome is the first among Pontibacillus that harbours a megaplasmid. Psychroflexus sp. ALD_RP9 is an aerobic heterotroph, which can generate energy by employing bacteriorhodopsins. It forms Gram-negative, orange, non-motile rods. The strain grows optimally at NaCl concentrations of 10%, pH 6.5–8 and 20°C. The Psychroflexus isolate tolerated pH conditions up to 10.5, which is the highest pH tolerance currently recorded for the genus. Psychroflexus sp. ALD_RP9 taxonomically belongs to the clade with the smallest genomes. Both isolates show extensive adaptations to their saline environments yet utilise different mechanisms to ensure survival.


Introduction
We describe two novel bacterial isolates from the Aldabra Atoll, Seychelles. They represent two novel species within the Pontibacillus and Psychroflexus genera. Samples from mangrove sediment within the lagoon (Pontibacillus isolate) and water from a moderately hypersaline pool on Grand Terre island (Psychroflexus isolate) were used for enrichment and isolation of halophilic bacteria.
The genus Pontibacillus belonging to the Bacillaceae was first described by Lim et al. as bacillus pertaining to the sea [1]. It harbours seven validated species, P. chungwhensis [1], P. halophilus [2], P. litoralis [3], P. marinus [4], P. salicampi [5], P. salipaludis [6], and P. yanchengensis [7]. Members of the Pontibacillus are Gram-positive, facultatively anaerobic, moderately halophilic, endospore-forming rods and are motile through peritrichous flagella. The isolates derive from marine-related habitats including salt farms across Asia and marine lifeforms, such as sea anemones and sea urchins [8]. They require salt for growth, which generally ranges from 0.5 to 25% (w/v) NaCl. Optimal growth was recorded between 2 and 10% (w/ v) NaCl. The pH optimum ranges from 7 to 8 and optimal growth temperatures from 25 to 40˚C. Members of Pontibacillus form white-to orange-pigmented smooth colonies with a diameter of 1 to 3 mm. The second isolate belongs to the genus Psychroflexus within the family Flavobacteriaceae. It was isolated from the pink, lower water layer of a saline landlocked pool (Westpool D). Psychroflexus (meaning cold bend) was first described by Bowman et al. in 1989 [9], who isolated the strain Ps. torquis from Antarctic sea ice and re-classified Flavobacterium gondwanense [10] to Ps. gondwanensis. The genus encompasses 12 validated and two non-validated published species. Psychroflexus species were isolated from hypersaline to saline lakes [11,12] and salterns in China [13], Hawaii [14], Korea [15,16], Antarctica [17], as well as saline soil [18], cheese [19] and coastal sediments [20,21]. Growth occurs between 0 and 20% (w/v) NaCl, a pH of 6 to 10, and -16 to 40˚C, demonstrating a high diversity in temperature tolerance and global dispersal of the genus. The growth optimum is generally around 2 to 10% NaCl, pH 7 to 8, and 10 to 15˚C or 25 to 30˚C, depending on arctic or tropical origin. Most isolates occur within the latter temperature range, thereby differing strongly from the type strain. All isolates of this genus are orange in colour.
In this study, we present two novel species affiliated to the Pontibacillus and the Psychroflexus genus. The isolates were characterised phenotypically (i) using standard microbiological techniques. In addition, complete genomes were generated using a hybrid approach of Illumina and Nanopore sequencing. Both genomes represent the second complete genome of the corresponding genus. The genomes were used to assess the phylogenetic affiliation (ii) and the potential metabolism (iii) of the strains.

Sampling, isolation and culture
Sampling and research permissions were granted by the Seychelles Islands Foundation (Research agreement: A26), the Seychelles Bureau of Standards (Research approval letter A0157) and the Ministry of Environment, Energy and Climate Change of the Republic of Seychelles (Non-commercial transfer of genetic materials agreement 4 th December 2017). Pontibacillus sp. ALD_SL1 and Psychroflexus sp. ALD_RP9 were isolated from halophile medium using mangrove sediment from the South Lagoon of the Aldabra Atoll (ALD_SL), Seychelles (9˚26'34.8''S, 46˚23'30.5''E) and water from the bottom water layer of Westpool D also known as Ronny's Pool (ALD_RP) (9˚26'40.5''S, 46˚27'6.8''E). The water (salinity 9.9%, pH 7.9, 0.10 mg/L O 2 , 32.5˚C) was sampled underneath a sharp halocline. The stratification of this pool was likely caused by the occurrence of the first rainfall, leading to a bacterial bloom in the sampled bottom layer. Prior to use, the untreated sediment was stored at -80˚C and the water sample at 4˚C. Liquid modified growth medium with 9% total salinity (MGM9) was prepared from 30% (w/v) concentrated saltwater stock solution (SW) as described by Dyall-Smith [22]. Briefly, the 30% SW was prepared from 240 g/L NaCl, 30 g/L MgCl 2 � 6 H 2 O, 35 g/L MgSO 4 7 H 2 O, 7 g/L KCl, 0.5 g/L CaCl 2 � 2 H 2 O and 0.2 g/L NaHCO 3 in deionized water. The pH was adjusted to 7.5 using tris(hydroxymethyl)aminomethane (Tris) buffer. For 1 L of MGM9, 300 mL of 30% SW were added to 5 g/L peptone (Oxoid) and 1 g/L yeast extract and deionized water. The pH was adjusted to 7.5 using Tris buffer. For solid medium, 15 g/L BactoAgar (BD Biosciences, Franklin Lakes, New Jersey, USA) was added before autoclaving. Untreated mangrove sediment (500 mg) was thawed, homogenized, and suspended in 1 ml of sterile 9% SW. Dilution series were prepared from the sediment suspension (SL) and from 100 μl of water sample (RP). They were plated on MGM9 and incubated at 28˚C in the light. Isolate ALD_SL1 was picked after two days from a sediment plate and ALD_RP9 was picked after five days from a plate with water sample. Both isolates were re-streaked at least three times to purify cultures. Pure isolates were stored at -80˚C in liquid MGM9 with 15% glycerol.

Morphology
Cells from a one-day-old culture were negatively stained with either 0.1% phosphotungstic acid (pH 7) or 0.5% uranyl acetate and applied onto a copper grid. Their morphology was determined using a Jeol 1011 electron microscope (Eching, Munich, Germany).

Growth experiments
Growth under differing NaCl, pH and temperature conditions were determined in an adjusted liquid MGM (nMGM). For this purpose, the SW was prepared without NaCl, which was added later in the required quantities. Salt tolerance was tested in 5% (w/v) increments of NaCl up to a concentration of 25% and 1% increments between 8 and 12%. For the determination of pH and temperature optima, the isolates were incubated in medium with 11% NaCl (Pontibacillus sp. ALD_SL1) and 9% NaCl (Psychroflexus sp. ALD_RP9). Temperature was tested between 10 and 50˚C at intervals of 5˚C between 20 and 40˚C and 28˚C instead of 30˚C, and pH values between 5.5 and 10.5 at intervals of 0.5. Pontibacillus sp. ALD_SL1 was incubated in 11% NaCl to reflect its slightly higher salt optimum. The pH intervals were adjusted with the addition of buffers (2-morpholinoethanesulfonic acid (MES), pH 5.5 and 6.0; Tris, pH 6.5 to 9.0; 3-(cyclohexylamino)-2-hydroxypropane-1-sulfonic acid (CAPSO), pH 9.5, 10.0 and 10.5) at concentrations of 1 M. For measurement of salinity and temperature optima, pH was set to 7.5. The pH and salinity experiments were incubated at 28˚C. All cultures were incubated using an Orbitron shaker (Infors HT, Einsbach, Germany) at 180 rpm. Growth under the different conditions was determined in triplicate (S1 Table). For this purpose, the optical density (OD 600 ) was measured using an Ultraspec 3300 Pro photometer (Amersham Pharmacia Biotec Europe GmbH, Munich, Germany) after 28 h, when both isolates had reached the stationary growth phase. Growth under anaerobic conditions was tested by placing inoculated nMGM plates with 11% NaCl (nMGM11) into an anaerobic jar with AnaeroGen 3.5 L gas packs (Thermo Fisher Scientific, Waltham, MA, USA) to generate an anaerobic atmosphere. The plates were incubated at 28˚C and monitored for colony growth for 14 days. Motility of the isolates was determined in soft nMGM11 with 3.5 g/L agar.

Physiological characterisation
Enzyme activity and carbohydrate utilisation of both isolates was tested using the API ZYM and API 50 CHB kits (bioMérieux, Nürtingen, Germany) according to the instructions of the manufacturer with adjusted salt concentrations. ALD_SL1 and ALD_RP9 cultures were washed twice before testing and resuspended in 11% or 5% saline respectively, for application in the API ZYM kit. The CHB medium of the 50 CHB kit was supplemented with 11% NaCl. Reactions in the 50 CHB kit were recorded up to 72 h of incubation. Oxidase production was tested by applying a drop of Oxidase Reagent (bioMérieux, Nürtingen, Germany) to a Rotilabo-test disk (; 6 mm). After adding a colony to the disk, it was monitored for a colour change. Catalase activity was tested in the same manner, but with 3% H 2 O 2 as reagent and monitoring for bubble development. Each test was replicated three times. Both isolates were examined by Gram-staining [23].

Genome sequencing
Genomic DNA was extracted from isolate cultures using the MasterPure Complete DNA and RNA Purification kit and the instructions of the manufacturer (Epicentre, Madison, USA) for the extraction of DNA from cell samples with the following adjustments. Cell cultures were pelleted and washed twice in PBS before DNA extraction. Cells were lysed in Cell Lysis Solution without Proteinase K and mechanically disrupted in a FastPrep (MP Biomedicals, Santa Ana, USA) for 20 s at 4 m/s with 0.1 mm glass beads. Afterwards, 2.5 μl of Proteinase K (20 mg/mL, Biotechrabbit, Düsseldorf, Germany) were added. Genomic DNA was eluted in 50 μl of nucleic acid free water and sequenced using both Illumina and Nanopore technology. Illumina paired-end reads were generated on a MiSeq sequencer using v3 chemistry (Illumina, San Diego, CA, USA) and Nanopore sequences were generated with a MinIon (Oxford Nanopore Technologies, Oxford, England) as described previously [24].

Bioinformatic processing and analysis
Illumina and Nanopore reads were quality-filtered using fastp v0.20.0 [25]. Nanopore longreads were filtered with a sequence cut-off of 1,000 bp (Psychroflexus sp. ALD_RP9) or 500 bp (Pontibacullus sp. ALD_SL1). Porechop v0.2.4 [26] was used for adapter-trimming and readsplitting. Sequences were assembled with Unicyler v0.4.8 and the conservative hybrid assembly approach [27]. Initial assessment of genome relatedness and taxonomy was performed using the Genome Taxonomy Database Tool kit (GTDB-Tk) and database v1.0.1 [28]. Assembled genomes were annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.13 [29] and are accessible under the accessions CP062974, CP062975 and CP062973. The 16S rRNA gene consensus sequences of each genome were aligned against the 16S rRNA gene sequences of all other members of each genus with ClustalW in MEGA-X 10.1.8 [30]. MEGA-X was also used to calculate neighbor-joining, maximum-likelihood and maximumparsimony phylogenetic trees with the Kimura two-parameter model and 1,000 bootstraps. Genome average nucleotide identity (ANI) was compared using the ANIm method in pyANI v0.2.10 [31] and similarities visualised using the Blast Ring Image Generator (BRIG) [32]. All available genome assemblies, ranging from contig via scaffold to complete, were used for ANI analysis. Cellular functions of both isolate genomes were inferred using BlastKOALA against the KEGG database [33] and pathway visualisation using the KEGG Mapper [34]. Transmembrane domains and signal peptides were predicted using TMHMM v.2.0 [35] and SignalP 5.0 servers [36]. Genomic islands and prophages were identified using IslandViewer4 [37] and PHASTER [38]. Putative antibiotic resistance genes (ARGs) were identified by searching the Resfams database v1.2.2 using HMMER 3.3 [39,40]. ARGs detected with Resfams were additionally verified with deepARG v2.0. Further hits were added to the putative list if identified genes crossed a threshold of 50% identity, a bit score above 50 and an e-value below 1e-20 [41].

Growth kinetics and optima
Both isolates were moderately halophilic with growth matching the reported ranges for members of the corresponding genus [5,12]. The NaCl optima were at 10% (ALD_RP9) and 11% (w/v) NaCl (ALD_SL1) ( Table 1, S1 Table). ALD_SL1 maintained growth between pH values of 6 and 10 with an optimum at pH 7. ALD_RP9 showed optimal growth between pH 6.5 and 8 and diminished growth between pH 9-10.5. Growth beyond pH 10.5 was not tested due to strong precipitation of medium components above this value. ALD_SL1 grew optimally at 28˚C. ALD_RP9 had a narrower growth range (20-40˚C) but same optimum as other members of the genus [19]. Incubation for 14 days under an anaerobic atmosphere showed growth of Pontibacillus sp. ALD_SL1, indicating that it is one of the facultative anaerobes of the genus [3,6]. Psychroflexus sp. ALD_RP9 showed only punctiform colonies, which may have benefited from residual oxygen at the start of the experiment.
Both isolates were tested for activity of certain enzymes and carbohydrate metabolism using API kits ZYM and CHB. Pontibacillus sp. ALD_SL1 was catalase positive and oxidase negative and showed enzyme activity for four of the 19 tested substrates, namely alkaline phosphatase, esterase (C4), esterase lipase (C8) and α-glucosidase activity. Acid production was observed for 10 of the 50 tested carbohydrates, including glycerol, D-ribose, D-glucose, Dfructose, D-maltose, sucrose, D-trehalose, inulin, starch, and glycogen (Table 1). This profile indicated that ALD_SL1 is metabolically more similar to P. chungwhensis than to P. salipaludis [6], which cluster together phylogenetically (Fig 2). Psychroflexus sp. ALD_RP9 was both catalase and oxidase negative and hydrolysed 9 of the 19 tested substrates using alkaline phosphatase, esterase (C4) and esterase lipase (C8), leucine, valine, and cysteine arylamidase, trypsin, acid phosphatase, and napthol-AS-BI-phosphohydrolase. Metabolic tests with the CHB kit showed acid production from maltose and starch (Table 1). While the growth ranges of all Psychroflexus isolates are similar, results from the metabolic tests differ strongly within the genus. Interpretation of these results is hampered by inconsistencies between studies and analyses regarding preparation (with/without NaCl) and incubation times (2-10 days) [12,19].

Genome assembly and characteristics
Two complete genomes were assembled with a hybrid assembly using long Nanopore reads and short Illumina paired-end reads. Quality-filtering with fastp removed 59% of the ALD_SL1 Nanopore reads, mainly due to the length constraints. Quality-filtering of ALD_SL1  (20) NaCl range (%, w/v) 5-20 (11) 5-15 (10) pH range 6-10 (7) Table 2). Of all sequenced Pontibacillus isolates, this is the only genome to harbour a (mega)plasmid. Of 1,236 hypothetical proteins in the whole genome, 725 are located on this plasmid. More than 50% of the megaplasmid were predicted as genomic islands, including two prophage regions. The chromosome harbours one putative prophage region (Fig 3). Quality filtering prior to Psychroflexus sp. ALD_RP9 assembly resulted in the removal of 16% of Nanopore and 4% of Illumina reads. The complete circular genome (CP062973) was assembled from 119,567 Nanopore reads with a mean length of 14,283 bp and 3,909,382 Illumina reads with a mean length of 262 bp. The genome comprises a single circular chromosome (2,555,922 bp) with a GC content of 33.1%. The mean read coverage was 1,056-fold. A total of 2,336 putative protein-encoding genes were assigned of which 431 were hypothetical. In addition, genes for nine rRNAs, 36 tRNAs and four non-coding RNAs were identified. Two genomic islands were identified, which overlap with the prophage predictions. These regions share low sequence similarity or are absent from the other Psychroflexus genomes (Fig 3).

Fig 2. Neighbor-joining phylogenetic tree of the Pontibacillus and Psychroflexus genera.
The tree includes 16S rRNA gene sequences from genomes ( � ), and genome scaffolds (S and scaffold number). The optimal tree with the sum of branch length 1.05278265 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. Bootstrapping values at the branches indicate the mean result of the neighbor-joining, maximum-likelihood and maximum-parsimony method. Evolutionary distances were calculated using the Kimura 2-parameter model with Archaeoglobus fulgidus L3 as an outgroup. https://doi.org/10.1371/journal.pone.0256639.g002

PLOS ONE
Two novel moderately halophilic bacterial isolates from the Aldabra Atoll

Phylogeny
A neighbour-joining phylogenetic tree was built with the consensus 16S rRNA gene sequences from each isolate and the 16S rRNA genes of all available genomes and scaffolds (Fig 2). GTDB-Tk resolved their taxonomy to the genus level with a RED value of 0.995 (ALD_SL1) and 0.994 (ALD_RP9), indicating that both are new species. Relatedness on the genomic level was assessed through average nucleotide identity analysis (ANIm) with all available genomes and genome assemblies (Fig 4). The available Pontibacillus genomes in the NCBI database consist of seven contig-level assemblies, two scaffolds and one complete genome (Pontibacillus sp. HMF3514). Pontibacillus sp. ALD_SL1 is most closely related to P. chungwhensis BH030062 T (ANI 93%) and P. salipaludis CGMCC 1.15353 (ANI 91%), which were isolated from a solar saltern in Chungwha, Korea [1] and marine sediment in Tuticorn, India [6]. All other available genome assemblies remain below 90% ANI, however, these values may be exaggerated due to gaps in the unclosed genomes and scaffolds. Available Psychroflexus assemblies comprise seven contig-level genomes, seven scaffolds and one complete genome (Ps. torquis). Psychroflexus sp. ALD_RP9 is most closely related to Ps. salarius MIC1008 T with an ANI of 89% and forms a well-supported cluster with Ps. saliphilus WDS4A14 T and Ps. halocasei WCC 4520 T . The relation of this branch to the cluster around the type species Ps. torquis ACAM627 T and Ps. tropicus LA1 T could not fully be resolved (bootstrap values < 60) using the three tree building methods (Fig 2). Members on this branch contain the smallest genomes of the genus with an average of 2.62 Mbp. Smaller genomes and a higher percentage of coding genes (92%) than more distant relatives i.e., Ps. torquis (4.32 Mbp, 81%) [42], indicate genome streamlining [43]. However, it has been proposed that the reverse is true and Ps. torquis may have benefitted from extensive gene acquisition to survive in its native arctic environment, leading to an increased genome size [42].
https://doi.org/10.1371/journal.pone.0256639.g003 to aminoglycosides, tetracycline, phosphomycin and ampicillin has previously been shown for other isolates of the genus [3,5,6]. ABC transporters not only convey resistance to antibiotics, but also act as channels for osmotic regulation in the marine environment. Transporters include Na + /H + antiporters (nhaC, mrpA-G) regulating cell homeostasis [44], K + transporters (trkA, kch), as well as mechanosensitive channels (mscL) (Fig 5A). Genes for the transport of compatible solutes proline, glycine and betaine (proV-X, opuD/betL, opuA-C), which act as osmolytes [45], were detected as in other Pontibacilli [8]. Genes for ectoine synthesis, which are common in many halotolerant bacteria [45 and references therein] as well as P. marinus [4], were not detected in the ALD_SL1 genome. Further regulation of osmotic and other stressors could occur via the encoded putative quorum-sensing and subsequent phosphorrelay systems. These are involved in the regulation of membrane fluidity (desK/R), degradative enzymes (degS/U) [46], competence (comX/K) [47], and induce sporulation (spo genes) [48] ( Fig 5A). While regulators of cell competence were present in the genome, a type IV secretion system for plasmid conjugation was not detected. ALD_SL1 can sense its environment and adjust its position using chemotaxis genes (mcp/che), which control flagella movement [49]. The chemotaxis receptors may be sensitive to oxygen, amongst other attractants, as the isolate showed aerotactic behaviour during motility testing. Most of its motility genes (i.e., fliG/M/N, motA/B) are situated both at the chromosome and the megaplasmid (Table 3). While encoding the same putative proteins, the genes share only low homology to each other. This indicates that they were acquired through horizontal gene transfer via the large mobile genetic elements (genomic islands/prophages) on the plasmid, rather than by interreplicon duplication [50]. As the plasmid does not harbour any essential genes, it is likely not critical for cell viability. Psychroflexus sp. ALD_RP9 genes matched 1,160 KEGG entries which were assigned to 214 pathways. Most entries belonged to amino acid (179) and carbohydrate (157) metabolism, followed by the metabolism of cofactors and vitamins (97). ARG search indicated that Psychroflexus sp. ALD_RP9 harbours six potential antibiotic resistance genes. Four of these are multidrug ABC transporters, one a multidrug and toxic compound extrusion (MATE) family resistance protein and one a bicyclomycin multidrug efflux protein (S2 Table). It has previously been shown, that Psychroflexi are resistant to aminoglycosides, polyketide, and quinolone antibiotics [13,20], but the identified ARGs cannot directly be linked to resistance against specific antibiotic compounds. Psychroflexus sp. ALD_RP9 employs a different strategy compared to Pontibacillus sp. ALD_SL1 to cope with the high salinity in its environment. To maintain turgor, it employs a series of Na + /H + antiporters (NhaC/D, MrpA) [44,51] and (mechanosensitive) ion transport channels (MscL/S, Kch) and proteins (TrkA) [52]. These may also support its ability to cope with the high pH tolerated. In addition, ALD_RP9 has an extensive exopolysaccharide layer surrounding the cells (Fig 1) and colonies have an almost jelly-like consistency. The EPS layer can protect from high salinity and pH values, as well as provide protection against desiccation [53]. ALD_RP9 forms its EPS layer by using a variety of nucleotide sugars and the Raetz pathway (lpx/waa genes). They are translocated and connected with the outer membrane via a lipopolysaccharide (LPS) transport system (lpt genes, Fig 5B) [54]. Acquisition of EPS/LPS genes has previously been observed in Ps. torquis, for which it was hypothesised that they support growth under psychrophilic conditions [42]. Psychroflexus sp. ALD_RP9 harbours some additional EPS genes within regions which are absent from all other strains (Fig 4). This suggests that ALD_RP9 has acquired additional EPS genes to support its survival. While EPS provides some desiccation protection, ALD_RP9 uses carotenoids (crtB/I/Y/Z genes) within its cell membrane to protect from irradiation and oxidative stress. The carotenoids can also be cleaved into retinal by Blh, which is required as co-factor for bacteriorhodopsin function [55]. In Psychroflexus members the bacteriorhodopsins most likely act as proton pumps that can drive ATP synthesis. This can be used to supplement their energy metabolism while under osmotic and other stress conditions [56,57].

Conclusions
We describe two novel bacterial species within the Pontibacillus and the Psychroflexus genus. Pontibacillus sp. ALD_SL1 is the third facultative anaerobe of the genus and first Pontibacillus to harbour a (mega-)plasmid. The plasmid contains genomic islands in large proportions and likely supports chemotaxis and motility but is not essential for cell viability. Psychroflexus sp. ALD_RP9 shows an enhanced capability to grow at high pH values in comparison to other Psychroflexus species. It clusters phylogenetically with members harbouring smallest genomes of the genus. Further, it contains genomic regions which are not present in other Psychroflexi and encode for additional genes involved in EPS synthesis. These may provide enhanced protection towards the moderately hypersaline conditions in its habitat. Taken together, both Metabolic pathways and capabilities were reconstructed from the complete genomes against the KEGG database using BlastKoala [33] and the KEGG Mapper [34]. https://doi.org/10.1371/journal.pone.0256639.g005

PLOS ONE
Pontibacillus sp. ALD_SL1 and Psychroflexus sp. ALD_RP9 are highly adapted to their environment but follow different strategies to support their survival.
Supporting information S1